Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation

Abstract

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolylhydroxylation in human hypoxia sensing has ancient origins.T he hypoxia-inducible transcription factor (HIF) is a major regulator of the response to limited oxygen availability in humans and other animals (1-3). A hypoxia-sensing component of the HIF system is provided by 2-oxoglutarate (2OG)-dependent and Fe(II)-dependent oxygenases, which catalyze prolyl-4-hydroxylation of HIF-α subunits, a posttranslational modification that enhances binding of HIF-α to the von Hippel-Lindau protein (pVHL), so targeting HIF-α for proteasomal degradation. The HIF prolyl-hydroxylases (PHDs) belong to a subfamily of 2OG oxygenases that catalyze prolyl-hydroxylation, which also includes the collagen prolyl-3-hydroxylases (CP3Hs) and prolyl-4-hydroxylases (CP4Hs) (4). Subsequently identified prolyl-hydroxylases include the ribosomal prolyl-hydroxylases (OGFOD1 and Tpa1), which catalyze ribosomal protein 23 prolyl-3-hydroxylation in many eukaryotes, and slime-mold enzymes, which catalyze prolyl-4-hydroxylation of Skp1, a ubiquitin ligase subunit (5-9). The HIF-PHD-VHL triad is likely present in all animals, but probably not in other organisms (3). However, structurally informed bioinformatic analyses imply the presence of PHD homologs in bacteria (10, 11), including in Pseudomonas spp, suggesting PHDs may have ancient origins. ResultsPseudomonas spp. Cont...

Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu

“…The structure of BaEFTu is not determined but exists as a monomer–dimer equilibrium in solution (Figure 4A, trace d). 27 The homology model of BaEFTu obtained from the crystal structure of P. putida EFTu (PDB entry 4JOQ) can be superimposed with the DAMMIF envelope for BaEFTu (Figures 3C and 6B). Theoretical scattering curves calculated using CRYSOL for BaEFTu and BaP4H dimer fit well ( χ 2 ∼ 1.6) with the experimental scattering profiles for the individual proteins (Figure 5D,E).…”

“…27 A crystal structure of the PPHD–EFTu complex shows conformational changes upon substrate binding and various substrate recognition interactions that are conserved among other P4Hs. 25,27 The sequences of neither EFTu nor TufB contain the canonical leucine-proline motifs or proline-rich repeats usually targeted by PHDs or P4Hs, respectively. Examination of the proteome of B. anthracis reveals the sequence of the highly conserved EFTu (BaEFTu) is ∼70% identical to those of the P. putida and S. oneidensis homologues.…”

OH, the Places You’ll Go! Hydroxylation, Gene Expression, and Cancer

“…Although these examples may simply be consistent with the requirement of 2OG oxygenase activity for Fe(II), they also highlight the potential for changes in Fe(II) availability to modulate hydroxylase activity. Consistent with this possibility, a prolyl hydroxylase in Pseudomonas has a relatively high K m for Fe(II) and is implicated in Fe(II) metabolism (Scotti et al, 2014). Ascorbate As discussed in the Introduction, the concept that loss of 2OG oxygenase activity due to nutrient deprivation could cause disease was illustrated by scurvy, where reduced collagen hydroxylase activity results from ascorbate deficiency.…”

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity

“…34 , 35 There are also two reported examples of bacterial prolyl 4-hydroxylases that form Hyp through posttranslational modification of peptidyl Pro. 36 , 37 Overall, it appears that eukaryotes are the primary source of Hyp with bacteria contributing to a much lesser extent.…”